Therapeutic agents 927

ABSTRACT

(2S)-2-[1-(2-Chloro-6-cyano-phenyl)pyrazolo[4,5-e]pyrimidin-4-yl]oxy-3-[(1R)-2-hydroxy-1-methyl-ethoxy]-N-(5-methylpyrazin-2-yl)propanamide or a pharmaceutically acceptable salt thereof is useful in the treatment or prevention of a disease or medical condition mediated through glucokinase (GLK or GK), leading to a decreased glucose threshold for insulin secretion.

This application claims the benefit under 35 U.S.C. §119(e) ofApplication No. 61/168,048 (US) filed on 9 Apr. 2009.

The present invention relates to(2S)-2-[1-(2-chloro-6-cyano-phenyl)pyrazolo[4,5-e]pyrimidin-4-yl]oxy-3-[(1R)-2-hydroxy-1-methyl-ethoxy]-N-(5-methylpyrazin-2-yl)propanamidewhich is useful in the treatment or prevention of a disease or medicalcondition mediated through glucokinase (GLK or GK). GK Activators (GKAs)are known to activate GK in the pancreatic β-cell leading to a decreasedglucose threshold for insulin secretion. In addition GKAs activatehepatic GK thereby stimulating hepatic glucose uptake and suppressinghepatic glucose output. The net pharmacological effect of GKAs is tolower blood glucose levels. Therefore, such compounds may have utilityin the treatment of Type 2 diabetes and obesity. The invention alsorelates to pharmaceutical compositions comprising said compounds and tomethods of treatment of diseases mediated by GLK using said compounds.

The biology of glucokinase and the mechanisms by which GKAs mightdeliver potential therapeutic benefit in Type 2 diabetes have beenextensively reviewed in the literature (see for example Matschinsky F Met al. (2006) Diabetes 55: 1-12, Leighton B, Atkinson A, Coghlan M P(2005) Biochemical Society Transactions 33: 371-374 and “Glucokinase andGlycemic Disease: From Basics to Novel Therapeutics.” Frontiers inDiabetes vol 16, eds. Matschinsky F M and Magnuson M A, Karger (Basel)2005). In the pancreatic β-cell and liver parenchymal cells the mainplasma membrane glucose transporter is GLUT2. Under physiologicalglucose concentrations the rate at which GLUT2 transports glucose acrossthe membrane is not rate limiting to the overall rate of glucose uptakein these cells. The rate of glucose uptake is limited by the rate ofphosphorylation of glucose to glucose-6-phosphate (G-6-P), which iscatalysed by glucokinase (GLK). GLK has a high (6-10 mM) Km for glucoseand is not inhibited by physiological concentrations of G-6-P. GLKexpression is limited to a few tissues and cell types, most notablypancreatic β-cells and liver cells (hepatocytes). In these cells GLKactivity is rate limiting for glucose utilisation and thereforeregulates the extent of glucose induced insulin secretion and hepaticglycogen synthesis. These processes are critical in the maintenance ofwhole body glucose homeostasis and both are dysfunctional in diabetes.

In one sub-type of diabetes, Maturity-Onset Diabetes of the Young Type 2(MODY-2), the diabetes is caused by GLK loss of function mutations.Hyperglycaemia in MODY-2 patients results from defective glucoseutilisation in both the pancreas and liver. Defective glucoseutilisation in the pancreas of MODY-2 patients results in a raisedthreshold for glucose stimulated insulin secretion. Conversely, rareactivating mutations of GLK reduce this threshold resulting in familialhyperinsulinism. In addition to the reduced GLK activity observed inMODY-2 diabetics, hepatic glucokinase activity is also decreased in type2 diabetics. Importantly, global or liver selective overexpression ofGLK prevents or reverses the development of the diabetic phenotype inboth dietary and genetic models of the disease. Moreover, acutetreatment of type 2 diabetics with fructose improves glucose tolerancethrough stimulation of hepatic glucose utilisation. This effect isbelieved to be mediated through a fructose induced increase in cytosolicGLK activity in the hepatocyte by the mechanism described below.

GLK and the K_(ATP) channel are expressed in neurones of thehypothalamus, a region of the brain that is important in the regulationof energy balance and the control of food intake. These neurones havebeen shown to express orectic and anorectic neuropeptides and have beenassumed to be the glucose-sensing neurones within the hypothalamus thatare either inhibited or excited by changes in ambient glucoseconcentrations. The ability of these neurones to sense changes inglucose levels is defective in a variety of genetic and experimentallyinduced models of obesity. Intracerebroventricular (icv) infusion ofglucose analogues, that are competitive inhibitors of glucokinase,stimulate food intake in lean rats. In contrast, icv infusion of glucosesuppresses feeding. Thus, small molecule activators of GLK may decreasefood intake and weight gain through central effects on GLK. Therefore,GLK activators may be of therapeutic use in treating eating disorders,including obesity, in addition to diabetes. The hypothalamic effectswill be additive or synergistic to the effects of the same compoundsacting in the liver and/or pancreas in normalising glucose homeostasis,for the treatment of Type 2 diabetes. Thus the GLK system can bedescribed as a potential “Diabesity” target (of benefit in both Diabetesand Obesity).

GLK is also expressed in specific entero-endocrine cells where it isbelieved to control the glucose sensitive secretion of the incretinpeptides GIP (glucose-dependent insulinotropic polypeptide) and GLP-1(Glucagon-Like Peptide-1) from gut K-cells and L-cells respectively.Therefore, small molecule activators of GLK may have additionalbeneficial effects on insulin secretion, b-cell function and survivaland body weight as a consequence of stimulating GIP and GLP-1 secretionfrom these entero-endocrine cells.

N-(Thiazolyl)-2-[(1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)thio]acetamideis disclosed in Phosphorus, Sulfur and Silicon, vol 178, 2003, pp1795-1805 which is concerned with antimicrobial activity of relatedcompounds. Similar compounds are also known from commercially availableChemical Libraries.

WO98/35944 discloses that compounds of formula R¹R²N—C(O)—C(R³)(R⁴)—X—R⁵in which R¹-R⁵ are each individually selected from a wide range ofsubstituents and X is oxygen or sulfur are useful in treating bulimiaand obesity by virtue of their activity at the NPY receptor antagonists.R⁵ may be 1H-pyrazolo[3,4-d]pyrimidin-4-yl or4-aminopyrazolo[3,4-d]pyrimidin-6-yl. Specific compounds exemplified areN-(4-cyclohexylphenyl)-2-(1H-pyrazolo[3,4-d]pyrimidin-4-ylsulfanyl)acetamide,N-(4-benzoylphenyl)-2-(1H-pyrazolo[3,4-d]pyrimidin-4-ylsulfanyl)acetamide,2-(4-amino-1H-pyrazolo[3,4-d]pyrimidin-6-ylsulfanyl)-N-(4-cyclohexylphenyl)acetamideand2-(4-amino-1H-pyrazolo[3,4-d]pyrimidin-6-ylsulfanyl)-N-(4-benzoylphenyl)acetamide.

Thus, according to the first aspect of the invention there is provided(2S)-2-[1-(2-chloro-6-cyano-phenyl)pyrazolo[4,5-e]pyrimidin-4-yl]oxy-3-[(1R)-2-hydroxy-1-methyl-ethoxy]-N-(5-methylpyrazin-2-yl)propanamideor a pharmaceutically acceptable salt thereof.

In particular the compound is(2S)-2-[1-(2-chloro-6-cyano-phenyl)pyrazolo[4,5-e]pyrimidin-4-yl]oxy-3-[(1R)-2-hydroxy-1-methyl-ethoxy]-N-(5-methylpyrazin-2-yl)propanamide.

It is to be understood that the compounds of the invention may exist intautomeric forms and that the invention also relates to any and alltautomeric forms of the compounds of the invention which activate GLK.It will be understood that compounds described herein in theirpyrimidinol form may also be described as the pyrimidinone tautomer andvice versa. This implies nothing about the actual relative proportionsof the two in physical samples.

It is also to be understood that certain compounds of the invention andsalts thereof can exist in solvated as well as unsolvated forms such as,for example, hydrated forms. It is to be understood that the inventionencompasses all such solvated forms which activate GLK.

In a further embodiment are provided in-vivo hydrolysable esters of thecompound of the invention, and in a further alternative embodiment areprovided pharmaceutically-acceptable salts of in-vivo hydrolysableesters of the compound of the invention.

The compound of the invention is named as apyrazolo[4,5-e]pyrimidin-4-yl compound. It will be understood by thoseskilled in the art that the compound could also be named as apyrazolo[3,4-d]pyrimidin-4-yl compound.

The compound of the invention may be administered in the form of apro-drug. A pro-drug is a bioprecursor or pharmaceutically acceptablecompound being degradable in the body to produce a compound of theinvention (such as an ester or amide of a compound of the invention,particularly an in-vivo hydrolysable ester). Various forms of prodrugsare known in the art. For examples of such prodrug derivatives, see:

a) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985) andMethods in Enzymology, Vol. 42, p. 309-396, edited by K. Widder, et al.(Academic Press, 1985);b) A Textbook of Drug Design and Development, edited byKrogsgaard-Larsen;

c) H. Bundgaard, Chapter 5 “Design and Application of Prodrugs”, by H.Bundgaard p. 113-191 (1991); d) H. Bundgaard, Advanced Drug DeliveryReviews, 8, 1-38 (1992); e) H. Bundgaard, et al., Journal ofPharmaceutical Sciences, 77, 285 (1988); and f) N. Kakeya, et al., ChemPharm Bull, 32, 692 (1984).

The contents of the above cited documents are incorporated herein byreference.

Examples of pro-drugs are as follows. An in-vivo hydrolysable ester of acompound of the invention containing a hydroxy group is, for example, apharmaceutically-acceptable ester which is hydrolysed in the human oranimal body to produce the parent alcohol. An in-vivo hydrolysable esterof a compound of the invention containing a hydroxy group includesinorganic esters such as phosphate esters (including phosphoramidiccyclic esters) and α-acyloxyalkyl ethers and related compounds which asa result of the in-vivo hydrolysis of the ester breakdown to give theparent hydroxy group. Examples of α-acyloxyalkyl ethers includeacetoxymethoxy and 2,2-dimethylpropionyloxy-methoxy. A selection ofin-vivo hydrolysable ester forming groups for hydroxy include alkanoyl,benzoyl, phenylacetyl and substituted benzoyl and phenylacetyl,alkoxycarbonyl (to give alkyl carbonate esters), dialkylcarbamoyl andN-(dialkylaminoethyl)-N-alkylcarbamoyl (to give carbamates),dialkylaminoacetyl and carboxyacetyl.

A suitable pharmaceutically-acceptable salt of a compound of theinvention is, for example, an acid-addition salt of a compound of theinvention which is sufficiently basic, for example, an acid-additionsalt with, for example, an inorganic or organic acid, for examplehydrochloric, hydrobromic, sulphuric, phosphoric, trifluoroacetic,citric or maleic acid. In one aspect, a suitable acid-addition salt maybe one with hydrochloric, sulphuric, methanesulfonic or citric acid.

A further feature of the invention is a pharmaceutical compositioncomprising the compound of the invention as defined above, or apharmaceutically-acceptable salt thereof, together with apharmaceutically-acceptable diluent or carrier.

According to another aspect of the invention there is provided thecompound of the invention or a pharmaceutically-acceptable salt thereoffor use as a medicament.

According to another aspect of the invention there is provided thecompound of the invention or a pharmaceutically-acceptable salt thereofas defined above for use as a medicament for treatment of a diseasemediated through GLK, in particular type 2 diabetes.

Further according to the invention there is provided the use of thecompound of the invention or a pharmaceutically-acceptable salt thereofin the preparation of a medicament for treatment of a disease mediatedthrough GLK, in particular type 2 diabetes.

The compound is suitably formulated as a pharmaceutical composition foruse in this way.

According to another aspect of the present invention there is provided amethod of treating GLK mediated diseases, especially diabetes, byadministering an effective amount of the compound of the invention or apharmaceutically-acceptable salt thereof to a mammal in need of suchtreatment.

Specific diseases which may be treated by a compound or composition ofthe invention include: blood glucose lowering in Type 2 DiabetesMellitus without a serious risk of hypoglycaemia (and potential to treattype 1), dyslipidemia, obesity, insulin resistance, metabolic syndromeX, impaired glucose tolerance.

As discussed above, thus the GLK system can be described as a potential“Diabesity” target (of benefit in both Diabetes and Obesity). Thus,according to another aspect of the invention there is provided the useof the compound of the invention or a pharmaceutically-acceptable saltthereof, in the preparation of a medicament for use in the combinedtreatment or prevention, particularly treatment, of diabetes andobesity.

According to another aspect of the invention there is provided the useof the compound of the invention or a pharmaceutically-acceptable saltthereof, in the preparation of a medicament for use in the treatment orprevention of obesity.

According to a further aspect of the invention there is provided amethod for the combined treatment of obesity and diabetes byadministering an effective amount of the compound of the invention or apharmaceutically-acceptable salt thereof, to a mammal in need of suchtreatment.

According to another aspect of the invention there is provided acompound the compound of the invention or a pharmaceutically-acceptablesalt thereof as defined above for use as a medicament for treatment orprevention, particularly treatment of obesity.

According to a further aspect of the invention there is provided amethod for the treatment of obesity by administering an effective amountof the compound of the invention or a pharmaceutically-acceptable saltthereof, to a mammal in need of such treatment.

Compounds of the invention may be particularly suitable for use aspharmaceuticals, for example because of favourable physical and/orpharmacokinetic properties and/or toxicity profile.

The compositions of the invention may be in a form suitable for oral use(for example as tablets, lozenges, hard or soft capsules, aqueous oroily suspensions, emulsions, dispersible powders or granules, syrups orelixirs), for topical use (for example as creams, ointments, gels, oraqueous or oily solutions or suspensions), for administration byinhalation (for example as a finely divided powder or a liquid aerosol),for administration by insufflation (for example as a finely dividedpowder) or for parenteral administration (for example as a sterileaqueous or oily solution for intravenous, subcutaneous, intramuscular orintramuscular dosing or as a suppository for rectal dosing). Dosageforms suitable for oral use are preferred.

The compositions of the invention may be obtained by conventionalprocedures using conventional pharmaceutical excipients, well known inthe art. Thus, compositions intended for oral use may contain, forexample, one or more colouring, sweetening, flavouring and/orpreservative agents.

Suitable pharmaceutically acceptable excipients for a tablet formulationinclude, for example, inert diluents such as lactose, sodium carbonate,calcium phosphate or calcium carbonate, granulating and disintegratingagents such as corn starch or algenic acid; binding agents such asstarch; lubricating agents such as magnesium stearate, stearic acid ortalc; preservative agents such as ethyl or propyl p-hydroxybenzoate, andanti-oxidants, such as ascorbic acid. Tablet formulations may beuncoated or coated either to modify their disintegration and thesubsequent absorption of the active ingredient within thegastrointestinal tract, or to improve their stability and/or appearance,in either case, using conventional coating agents and procedures wellknown in the art.

Compositions for oral use may be in the form of hard gelatin capsules inwhich the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules in which the active ingredient is mixed with water oran oil such as peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions generally contain the active ingredient in finelypowdered form together with one or more suspending agents, such assodium carboxymethylcellulose, methylcellulose,hydroxypropylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone,gum tragacanth and gum acacia; dispersing or wetting agents such aslecithin or condensation products of an alkylene oxide with fatty acids(for example polyoxethylene stearate), or condensation products ofethylene oxide with long chain aliphatic alcohols, for exampleheptadecaethyleneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol such aspolyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with long chain aliphatic alcohols, for exampleheptadecaethyleneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol such aspolyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides, for example polyethylene sorbitan monooleate. The aqueoussuspensions may also contain one or more preservatives (such as ethyl orpropyl p-hydroxybenzoate, anti-oxidants (such as ascorbic acid),colouring agents, flavouring agents, and/or sweetening agents (such assucrose, saccharine or aspartame).

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil (such as arachis oil, olive oil, sesame oil orcoconut oil) or in a mineral oil (such as liquid paraffin). The oilysuspensions may also contain a thickening agent such as beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set outabove, and flavouring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of ananti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water generally contain the activeingredient together with a dispersing or wetting agent, suspending agentand one or more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients such as sweetening, flavouring and colouringagents, may also be present.

The pharmaceutical compositions of the invention may also be in the formof oil-in-water emulsions. The oily phase may be a vegetable oil, suchas olive oil or arachis oil, or a mineral oil, such as for exampleliquid paraffin or a mixture of any of these. Suitable emulsifyingagents may be, for example, naturally-occurring gums such as gum acaciaor gum tragacanth, naturally-occurring phosphatides such as soya bean,lecithin, an esters or partial esters derived from fatty acids andhexitol anhydrides (for example sorbitan monooleate) and condensationproducts of the said partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate. The emulsions may also containsweetening, flavouring and preservative agents.

Syrups and elixirs may be formulated with sweetening agents such asglycerol, propylene glycol, sorbitol, aspartame or sucrose, and may alsocontain a demulcent, preservative, flavouring and/or colouring agent.

The pharmaceutical compositions may also be in the form of a sterileinjectable aqueous or oily suspension, which may be formulated accordingto known procedures using one or more of the appropriate dispersing orwetting agents and suspending agents, which have been mentioned above. Asterile injectable preparation may also be a sterile injectable solutionor suspension in a non-toxic parenterally-acceptable diluent or solvent,for example a solution in 1,3-butanediol.

Compositions for administration by inhalation may be in the form of aconventional pressurised aerosol arranged to dispense the activeingredient either as an aerosol containing finely divided solid orliquid droplets. Conventional aerosol propellants such as volatilefluorinated hydrocarbons or hydrocarbons may be used and the aerosoldevice is conveniently arranged to dispense a metered quantity of activeingredient.

For further information on formulation the reader is referred to Chapter25.2 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch;Chairman of Editorial Board), Pergamon Press 1990.

The amount of active ingredient that is combined with one or moreexcipients to produce a single dosage form will necessarily varydepending upon the host treated and the particular route ofadministration. For example, a formulation intended for oraladministration to humans will generally contain, for example, from 0.5mg to 2 g of active agent compounded with an appropriate and convenientamount of excipients which may vary from about 5 to about 98 percent byweight of the total composition. Dosage unit forms will generallycontain about 1 mg to about 500 mg of an active ingredient. For furtherinformation on Routes of Administration and Dosage Regimes the reader isreferred to Chapter 25.3 in Volume 5 of Comprehensive MedicinalChemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press1990.

The size of the dose for therapeutic or prophylactic purposes of acompound of the compound of the invention will naturally vary accordingto the nature and severity of the conditions, the age and sex of theanimal or patient and the route of administration, according to wellknown principles of medicine.

In using a compound of the invention for therapeutic or prophylacticpurposes it will generally be administered so that a daily dose in therange, for example, 0.5 mg to 75 mg per kg body weight is received,given if required in divided doses. In general lower doses will beadministered when a parenteral route is employed. Thus, for example, forintravenous administration, a dose in the range, for example, 0.5 mg to30 mg per kg body weight will generally be used. Similarly, foradministration by inhalation, a dose in the range, for example, 0.5 mgto 25 mg per kg body weight will be used. Oral administration is howeverpreferred.

The elevation of GLK activity described herein may be applied as a soletherapy or in combination with one or more other substances and/ortreatments for the indication being treated. In another aspect theinvention provides a pharmaceutical combination comprising a compound ofthe invention and another pharmacologically active substanceparticularly wherein the other pharmacologically active substance is amedicament for the treatment of type 2 diabetes or obesity or a relatedcondition.

Such conjoint treatment may be achieved by way of the simultaneous,sequential or separate administration of the individual components ofthe treatment. Simultaneous treatment may be in a single tablet or inseparate tablets. For example in the treatment of diabetes mellitus,chemotherapy may include the following main categories of treatment:

-   -   1) Insulin and insulin analogues;    -   2) Insulin secretagogues including sulphonylureas (for example        glibenclamide, glipizide), prandial glucose regulators (for        example repaglinide, nateglinide);    -   3) Agents that improve incretin action (for example dipeptidyl        peptidase IV inhibitors e.g. saxagliptin, sitagliptin,        vildagliptin or alogliptin and GLP-1 agonists);    -   4) Insulin sensitising agents including PPARgamma agonists (for        example pioglitazone and rosiglitazone), and agents with        combined PPARalpha and gamma activity;    -   5) Agents that modulate hepatic glucose balance (for example        metformin, fructose 1, 6 bisphosphatase inhibitors, glycogen        phosphorylase inhibitors, glycogen synthase kinase inhibitors);    -   6) Agents designed to reduce the absorption of glucose from the        intestine (for example acarbose);    -   7) Agents that prevent the reabsorption of glucose by the kidney        (SGLT inhibitors for example dapagliflozin);    -   8) Agents designed to treat the complications of prolonged        hyperglycaemia (for example aldose reductase inhibitors);    -   9) Anti-obesity agents (for example sibutramine and orlistat);    -   10) Anti-dyslipidaemia agents such as, HMG-CoA reductase        inhibitors (eg statins); PPARα agonists (fibrates, eg        gemfibrozil); bile acid sequestrants (cholestyramine);        cholesterol absorption inhibitors (plant stanols, synthetic        inhibitors); bile acid absorption inhibitors (IBATi) and        nicotinic acid and analogues (niacin and slow release        formulations);    -   11) Antihypertensive agents such as, β blockers (eg atenolol,        inderal); ACE inhibitors (eg lisinopril); Calcium antagonists        (eg. nifedipine); Angiotensin receptor antagonists (eg        candesartan), α antagonists and diuretic agents (eg. furosemide,        benzthiazide);    -   12) Haemostasis modulators such as, antithrombotics, activators        of fibrinolysis and antiplatelet agents; thrombin antagonists;        factor Xa inhibitors; factor VIIa inhibitors); antiplatelet        agents (eg. aspirin, clopidogrel); anticoagulants (heparin and        Low molecular weight analogues, hirudin) and warfarin;    -   13) Agents which antagonise the actions of glucagon; and    -   14) Anti-inflammatory agents, such as non-steroidal        anti-inflammatory drugs (eg. aspirin) and steroidal        anti-inflammatory agents (eg. cortisone).        The compound of the invention, or a salt thereof, may be        prepared by any process known to be applicable to the        preparation of such compounds or structurally related compounds.        Functional groups may be protected and deprotected using        conventional methods. For examples of protecting groups such as        amino and carboxylic acid protecting groups (as well as means of        formation and eventual deprotection), see T. W. Greene        and P. G. M. Wuts, “Protective Groups in Organic Synthesis”,        Second Edition, John Wiley & Sons, New York, 1991.

Processes for the synthesis of the compound of the invention areprovided as a further feature of the invention. Thus, according to afurther aspect of the invention there is provided a process for thepreparation of a compound of the invention as described in the examples.

In a further aspect the present invention provides a process for thepreparation of(2S)-2-[1-(2-chloro-6-cyano-phenyl)pyrazolo[4,5-e]pyrimidin-4-yl]oxy-3-[(1R)-2-hydroxy-1-methyl-ethoxy]-N-(5-methylpyrazin-2-yl)propanamidecomprising de-protecting a compound of formula A

in which Prot represents a hydroxy protecting group.

Examples of hydroxy protecting groups include methyl, t-butyl, loweralkenyl groups (e.g. allyl); lower alkanoyl groups (e.g. acetyl); loweralkoxycarbonyl groups (e.g. t-butoxycarbonyl); lower alkenyloxycarbonylgroups (e.g. allyloxycarbonyl); aryl lower alkoxycarbonyl groups (e.g.benzoyloxycarbonyl, p-methoxybenzyloxycarbonyl,o-nitrobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl); tri loweralkyl/arylsilyl groups (e.g. trimethylsilyl, t-butyldimethylsilyl,t-butyldiphenylsilyl); tetrahydropyran-2-yl; aryl lower alkyl groups(e.g. benzyl) groups; and triaryl lower alkyl groups (e.g.triphenylmethyl).

Methods appropriate for removal of hydroxy protecting groups include,for example, nucleophilic displacement, acid-, base-, metal- orenzymically-catalysed hydrolysis, catalytic hydrogenolysis/hydrogenationor photolytically for groups such as o-nitrobenzyloxycarbonyl. Silylgroups may be removed with fluoride ions for example usingtetrabutylammonium fluoride in a solvent, for example an ether e.g.tetrahydrofuran or an alkanoic acid for example acetic acid, at atemperature from 0° C. to the boiling point of the solvent or moreparticularly in the range 5° C.-35° C. Silyl groups may be also removedwith a mineral acid for example hydrochloric acid, particularly dilutehydrochloric acid, at a temperature from 0° C. to 50° C., particularly15-30° C. Silyl groups may be removed with organic acids e.galkylsulfonic acids, for example methanesulfonic acid, or arylsulfonicacids, for example benzenesulfonic acid. Methylether protecting groupsfor hydroxy groups may be removed by trimethylsilyliodide. A tert-butylether protecting group for a hydroxy group may be removed by treatmentwith an acid, for example by use of hydrochloric acid in methanol.

Certain intermediates used to prepare the compound of the invention arebelieved to be novel and are herein claimed as a further embodiment ofthe present invention. In another aspect the present invention providesa compound of formula A. Particularly Prot represents a tri loweralkyl/arylsilyl groups (e.g. trimethylsilyl, t-butyldimethylsilyl,t-butyldiphenylsilyl).

In a further embodiment the present invention provides one or more ofthe following:

-   (2S)-2-(1-(2-chloro-6-cyanophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)-N-(5-methylpyrazin-2-yl)-3-((R)-1-(triisopropylsilyloxy)propan-2-yloxy)propanamide;-   (S)-2-hydroxy-N-(5-methylpyrazin-2-yl)-3-((R)-1-(triisopropylsilyloxy)propan-2-yloxy)propanamide;    and-   (2S)-2-[1-(2-bromo-6-chlorophenyl)pyrazolo[4,5-e]pyrimidin-4-yl]oxy-N-(5-methylpyrazin-2-yl)-3-[(2R)-1-tri(propan-2-yl)silyloxypropan-2-yl]oxypropanamide.    The following examples are for illustration purposes and are not    intended to limit the scope of this application. Each exemplified    compound represents a particular and independent aspect of the    invention. In the following non-limiting Examples, unless otherwise    stated:

(i) evaporations were carried out by rotary evaporation under reducedpressure and work-up procedures were carried out after removal ofresidual solids such as drying agents by filtration;

(ii) operations were carried out at room temperature, that is in therange 18-25° C. and under an atmosphere of an inert gas such as argon ornitrogen;

(iii) yields are given for illustration only and are not necessarily themaximum attainable;

(iv) the structures of the end-products of the Examples were confirmedby nuclear (generally proton) magnetic resonance (NMR) and mass spectraltechniques; proton magnetic resonance chemical shift values weremeasured on the delta scale and peak multiplicities are shown asfollows: s, singlet; d, doublet; t, triplet; m, multiplet; br, broad; q,quartet; quin, quintet; sextet

(v) intermediates were not generally fully characterised and purity wasassessed by thin layer chromatography (TLC), high-performance liquidchromatography (HPLC), infra-red (IR) or NMR analysis;

(vi) flash chromatography was carried out on silica unless otherwisestated;

(vii) Enantiomeric excesses (ee's) were determined by HPLC using achiral stationary phase such as Chiralcel OJ or Chiralpak AD-H and/or byNMR using an appropriate chiral shift reagent such as(1S)-[1,1′-binaphthalene]-2,2′-diol (CAS 18531-99-2) or(1R)-[1,1′-binaphthalene]-2,2′-diol (CAS 18531-94-7).

ABBREVIATIONS

-   ACN Acetonitrile-   n-BuLi n-Butyllithium-   m-CPBA 3-Chloroperbenzoic acid-   DCM Dichloromethane-   DIPEA Di-iso-propylethylamine-   DMAP 4-Di(methylamino)pyridine-   DMF N,N-Dimethylformamide-   DMSO dimethylsulfoxide-   EtOAc Ethyl acetate-   EtOH Ethanol-   FMOC 9-Fluorenylmethyl carbamate-   IPA Isopropyl alcohol-   LHMDS Lithium bis(trimethylsilyl)amide-   MeOH Methanol-   TBDMSCl tert-Butyldimethylsilyl chloride-   THF Tetrahydrofuran-   ESI Electrospray ionisation-   rt Room temperature-   cat Catalytic-   ee Enantiomeric excess-   HPLC High performance liquid chromatography-   EDCI 1-(3-Dimethylaminopropyl)-2-ethylcarbodiimide hydrochloride-   rel vols relative volumes-   equiv molar equivalents

EXAMPLE 1(2S)-2-[1-(2-chloro-6-cyano-phenyl)pyrazolo[4,5-e]pyrimidin-4-yl]oxy-3-[(1R)-2-hydroxy-1-methyl-ethoxy]-N-(5-methylpyrazin-2-yl)propanamide

A solution of tetrabutylammonium fluoride (1M in THF) (0.887 mL, 0.89mmol) was added in one portion to a stirred solution of(2S)-2-(1-(2-chloro-6-cyanophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)-N-(5-methylpyrazin-2-yl)-3-((R)-1-(triisopropylsilyloxy)propan-2-yloxy)propanamide(Intermediate AZ1) (0.59 g, 0.89 mmol) in tetrahydrofuran (15 mL). Theresulting solution was stirred at ambient temperature for 30 minutes.The reaction mixture was quenched with saturated NH₄Cl (10 mL), anddiluted with water (20 mL) and EtOAc (70 mL). The organic layer wasseparated and the aqueous layer extracted with EtOAc (70 mL), Thecombined organics were dried (MgSO₄), filtered and evaporated to affordcrude product. The crude product was purified by flash silicachromatography, elution gradient 50 to 80% EtOAc in isohexane to affordthe product (0.367 g, 81%). ¹H NMR (400 MHz, CDCl3) δ 1.14-1.20 (3H, m),2.54 (3H, s), 2.70-2.84 (1H, m), 3.54-3.72 (2H, m), 3.77-3.83 (1H, m),4.09-4.16 (1H, m), 4.28-4.32 (1H, m), 6.08-6.11 (1H, m), 7.63 (1H, t),7.78-7.81 (1H, m), 7.85-7.88 (1H, m), 8.12 (1H, s), 8.50-8.53 (1H, m),8.62 (1H, d), 9.04-9.12 (1H, m), 9.41 (1H, s); m/z (ES⁺) (M+H)⁺=509.06;HPLC t_(R)=2.41 min.

(2S)-2-(1-(2-chloro-6-cyanophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)-3-((R)-1-hydroxypropan-2-yloxy)-N-(5-methylpyrazin-2-yl)propanamide(220 mg, 0.43 mmol) was taken up into Et₂O (20 mL) and warmed to boilingunder reflux. The material dissolved and then began to crystallise. Themixture was allowed to cool and stood at ambient temperature for 1 hour.The solid was filtered off to give(2S)-2-(1-(2-chloro-6-cyanophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)-3-((R)-1-hydroxypropan-2-yloxy)-N-(5-methylpyrazin-2-yl)propanamide(200 mg) which was shown to be crystalline by x-ray powder diffraction.

Angle 2- Theta (2θ) Intensity % 4.234 100.0 20.902 95.6 14.456 95.217.730 77.8 13.278 69.4 11.065 63.2 21.237 61.5 19.725 59.4 26.458 59.28.463 54.9 26.192 54.8

EXAMPLE 1(2S)-2-[1-(2-chloro-6-cyano-phenyl)pyrazolo[4,5-e]pyrimidin-4-yl]oxy-3-[(1R)-2-hydroxy-1-methyl-ethoxy]-N-(5-methylpyrazin-2-yl)propanamideAlternative method

Tetrabutylammonium fluoride 1M in THF (5.11 mL, 5.11 mmol) was added to(2S)-2-(1-(2-chloro-6-cyanophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)-N-(5-methylpyrazin-2-yl)-3-((R)-1-(triisopropylsilyloxy)propan-2-yloxy)propanamide(Intermediate AZ1) (3.4 g, 5.11 mmol) and acetic acid (0.307 g, 5.11mmol) in THF (35 mL). The resulting solution was stirred at 22° C. for20 hours. It was quenched with NH₄Cl solution (sat., 25 mL) and dilutedwith EtOAc (50 mL). The organic phase was washed with saturated sodiumbicarbonate (25 mL), water (20 mL), brine (20 mL), dried (MgSO₄) andevaporated. 6 The crude product was purified by flash silicachromatography, eluting with EtOAc to afford the product (2.23 g, 85%).This was crystallised from EtOAc (10 mL) and iso-hexane (6 mL) to givethe desired product as a white crystalline powder (1.9 g, 73%) confirmedto be of the same crystalline form as that described previously.

¹H NMR (400 MHz, DMSO-d₆) δ 1.06-1.07 (3H, m), 2.44 (3H, s), 3.25-3.47(2H, m), 3.62-3.67 (1H, m), 4.09-4.16 (2H, m), 4.53-4.61 (1H, m),5.90-5.96 (1H, m), 7.89 (1H, dd), 8.16-8.20 (2H, m), 8.31-8.32 (1H, m),8.62 (1H, s), 8.76-8.78 (1H, m), 9.10-9.12 (1H, m), 11.17 (1H, s); m/z(ES⁻) (M−H)⁻=507; HPLC t_(R)=2.44 min.

EXAMPLE 1(2S)-2-[1-(2-chloro-6-cyano-phenyl)pyrazolo[4,5-e]pyrimidin-4-yl]oxy-3-[(1R)-2-hydroxy-1-methyl-ethoxy]-N-(5-methylpyrazin-2-yl)propanamideAlternative method

Hydrochloric acid (6M, 4 equivs) was added with stirring to a solutionof(2S)-2-(1-(2-chloro-6-cyanophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)-N-(5-methylpyrazin-2-yl)-3-((R)-1-(triisopropylsilyloxy)propan-2-yloxy)propanamide(Intermediate AZ1) (1 equiv) in propan-2-ol (9 rel vols) in an inertatmosphere at ambient temperature. The mixture was stirred for 6 hoursthen basified carefully with aqueous sodium bicarbonate solution. Thevolume was reduced to half of the original volume by distillation underreduced pressure and then ethyl acetate and water were added. Themixture was stirred and then the phases were separated. The aqueousphase was extracted with ethyl acetate and the combined organic extractand washings were dried by azeotropic distillation. The product wascrystallised from a mixture of ethyl acetate/heptane (5:6 vols). Yield84%.

Preparation of Intermediates Intermediate AH1: di-tert-butyl1-(2-bromo-6-chlorophenyl)hydrazine-1,2-dicarboxylate

To a stirred solution of 2.5 M Butyllithium in hexanes (13.79 mL, 34.47mmol) at −20° C. was added dropwise 2,2,6,6-tetramethylpiperidine (5.82mL, 34.47 mmol) in THF (30 mL). This was stirred for 30 minutes. Thissolution was cooled down to −78° C., and a solution of1-bromo-3-chlorobenzene (6 g, 31.34 mmol) in THF (20 mL) was addeddropwise so as to maintain the temperature at <−70° C. After 2 hours(Z)-di-tert-butyl diazene-1,2-dicarboxylate (10.82 g, 47.01 mmol) in THF(30 mL) added dropwise at −78° C. Stirred at this temperature for 2hours, then allowed to warm to room temperature overnight. Water (150mL) added. The mixture was extracted with EtOAc (2×300 mL). The extractswere combined, washed with brine (100 mL), dried (MgSO₄) and reduced gogive a residue which was purified by flash silica chromatography,elution gradient 0-10% EtOAc in hexane. Fractions containing suspectedproduct were combined, and reduced to give di-tert-butyl1-(2-bromo-6-chlorophenyl)hydrazine-1,2-dicarboxylate (9.59 g, 72.5%) ¹HNMR (400 MHz, DMSO) δ 1.30-1.50 (18H, s), 7.28 (1H, m), 7.55 (1H, dd),7.67 (1H, dd), 9.18 (1H, s). m/z (ES+) (M+Na)⁺=443; HPLC t_(R)=10.76min.

Intermediate AH2: (2-bromo-6-chlorophenyl)hydrazine hydrochloride

4M Hydrogen chloride in dioxane (28.2 mL, 112.64 mmol) was added todi-tert-butyl 1-(2-bromo-6-chlorophenyl)hydrazine-1,2-dicarboxylate(Intermediate AH1) (9.5 g, 22.53 mmol) in iPrOH (30 mL) at ambienttemperature. The resulting solution was heated to 60° C. and stirred for20 minutes. The reaction mixture was allowed to cool to ambienttemperature. A solid was filtered off, and dried under vacuum to affordthe product (4.71 g). ¹H NMR (400 MHz, DMSO) d 6.97 (1H, s), 7.06 (1H,t), 7.41 (1H, dd), 7.52 (1H, dd), 9.83 (3H, s). m/z M⁺=220; GC-MStR=10.419 min.

Intermediate AH3:5-amino-1-(2-bromo-6-chlorophenyl)-1H-pyrazole-4-carbonitrile

(2-Bromo-6-chlorophenyl)hydrazine hydrochloride (Intermediate AH2) (4.71g, 18.26 mmol) was partitioned between DCM (300 mL) and 2M NaOH (20 mL).Allowed to Stir for 1 hour. The two phases were separated. The organicphase was reduced under vacuum to give the free base. This was dissolvedin MeOH (60 mL), and stirred in a cooling bath at 0° C.(Ethoxymethylene)malononitrile (2.230 g, 18.26 mmol) added portionwise.The reaction mixture was then allowed to warm to ambient temperature andstirred for 1 hour. The mixture was then heated to reflux and stirredfor 16 hours. Allowed to cool to ambient temperature. The reactionmixture was concentrated to give a solid, which was triturated underMeOH. A solid was filtered off. This was dried to afford the product(4.59 g, 85%). ¹H NMR (400 MHz, DMSO) δ 6.79 (2H, s), 7.54-7.47 (1H, m),7.70 (1H, dd), 7.79 (1H, s), 7.81 (1H, d). m/z (ES+) (M+H)⁺=299; HPLCt_(R)=1.70 min.

Intermediate AH4:1-(2-bromo-6-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one

Concentrated sulfuric acid (0.905 mL, 16.97 mmol) was added to a stirredsolution of5-amino-1-(2-bromo-6-chlorophenyl)-1H-pyrazole-4-carbonitrile(Intermediate AH3) (4.59 g, 15.43 mmol) in formic acid (30 mL). Theresulting solution was heated to 100° C. and stirred for 4 hours. Thereaction mixture was allowed to cool to ambient temperature. Thereaction mixture was evaporated to dryness. The residue was trituratedunder water. A solid was filtered off. This was dried under vacuum toafford the product (3.71 g, 73.8%). ¹H NMR (400 MHz, DMSO) δ 7.58 (1H,t), 7.80-7.76 (1H, m), 7.88 (1H, dd), 8.08 (1H, d), 8.40 (1H, s), 12.44(1H, s). m/z (ES+) (M+H)⁺=327; HPLC t_(R)=1.48 min.

Intermediate AH5:3-chloro-2-(4-hydroxy-1H-pyrazolo[3,4-d]pyrimidin-1-yl)benzonitrile

1-(2-bromo-6-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one(Intermediate AH4) (1012 mg, 3.11 mmol) and zinc cyanide (329 mg, 2.80mmol) were dissolved in DMF (15 mL). Sealed into a microwave tube. Thetube was purged with nitrogen.9,9-Dimethyl-4,5-bis(diphenylphosphino)xanthene (180 mg, 0.31 mmol) andtris(dibenzylideneacetone)dipalladium(0) (142 mg, 0.16 mmol) added. Thereaction was heated to 160° C. for 15 minutes in the microwave reactor,then allowed to cool to ambient temperature. The DMF was removed byevaporation. The crude product was absorbed onto silica and thenpurified by flash silica chromatography, elution gradient 0 to 10% MeOHin DCM to afford the product (769 mg, 91%). ¹H NMR (400 MHz, DMSO) δ7.89-7.83 (1H, t), 8.14 (1H, s), 8.15 (2H, d), 8.49 (1H, s), 12.56 (1H,s); m/z (ES+) (M+H)⁺=272; HPLC t_(R)=1.29 min.

Intermediate AH6:3-Chloro-2-(4-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)benzonitrile

Phosphorus oxychloride (7.93 mL, 85.03 mmol) was added to3-chloro-2-(4-hydroxy-1H-pyrazolo[3,4-d]pyrimidin-1-yl)benzonitrile(Intermediate AH5) (1.155 g, 4.25 mmol). The resulting mixture washeated to 100° C. and stirred for 4 hours. The reaction mixture wasallowed to cool to ambient temperature. The reaction mixture wasevaporated to near dryness. Poured onto ice/water with stirring. A solidprecipitate was collected by filtration. This was dried overnight undervacuum to afford the product (0.928 g, 75%). ¹H NMR (400 MHz, DMSO) δ8.02-7.96 (1H, t), 8.26 (1H, d), 8.28 (1H, s), 9.05 (2H, d). m/z M⁺=289;GCMS tR=13.82 min.

Intermediate AN1: (S)-Methyl3-((R)-1-(tert-butyldimethylsilyloxy)propan-2-yloxy)-2-hydroxypropanoate

Magnesium trifluoromethanesulfonate (1.165 g, 3.61 mmol) was added inone portion to (S)-methyl oxirane-2-carboxylate (1.475 g, 14.45 mmol)and (R)-1-(tert-butyldimethylsilyloxy)propan-2-ol (CAS no. 136918-07-5)(2.75 g, 14.45 mmol) cooled to 10° C. The resulting suspension wasstirred at 10° C. for 10 minutes and then warmed to 45° C. and stirredfor 2 days. The crude product was purified by flash silicachromatography, eluting with 0 to 30% EtOAc in isohexane to afford theproduct (1.680 g, 39.8%). ¹H NMR (400 MHz, DMSO) δ0.00 (s, 6H), 0.83 (s,9H), 0.98 (d, 3H), 3.35-3.45 (m, 2H), 3.47-3.65 (m, 3H), 3.66 (s, 3H),4.09-4.15 (m, 1H), 5.37 (d, 1H).

Intermediate AU5: (S)-methyl2-hydroxy-3-((R)-1-(triisopropylsilyloxy)propan-2-yloxy)propanoate

Prepared using a procedure analogous to that described for IntermediateAN1 using (R)-1-(triisopropylsilyloxy)propan-2-ol (CAS no. 871657-72-6).¹H NMR (400 MHz, CDCl₃) δ 1.04-1.16 (24H, m), 3.39 (1H, d), 3.54-3.59(2H, m), 3.67-3.72 (1H, m), 3.78 (3H, s), 3.80-3.91 (2H, m), 4.27-4.31(1H, m).

Intermediate AU5: (R)-methyl2-hydroxy-3-((S)-1-(triisopropylsilyloxy)propan-2-yloxy)propanoateAlternative Procedure

Magnesium trifluoromethanesulfonate (1.263 g, 3.92 mmol) was added to amixture of (S)-1-(triisopropylsilyloxy)propan-2-ol (4.55 g, 19.59 mmol)and (R)-methyl oxirane-2-carboxylate (2 g, 19.59 mmol) in ethyl acetate(40 mL). The resulting suspension was heated to 80° C. for 72 hours.Further (R)-methyl oxirane-2-carboxylate (2 g, 19.59 mmol) was added andthe mixture stirred at 80° C. for 48 hrs. Further (R)-methyloxirane-2-carboxylate (2 g, 19.59 mmol) was added and the mixturestirred for 24 hrs. The mixture was allowed to cool, filtered through abed of silica (˜10 g), washed through with EtOAc and evaporated. Thecrude product was purified by flash silica chromatography, elutiongradient 10 to 50% EtOAc in isohexane, to afford the product (2.7 g,41%).

Intermediate AZ1:(2S)-2-(1-(2-chloro-6-cyanophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)-N-(5-methylpyrazin-2-yl)-3-((R)-1-(triisopropylsilyloxy)propan-2-yloxy)propanamide

Sodium hydride (60% dispersion in mineral oil) (0.116 g, 2.89 mmol) wasadded to(S)-2-hydroxy-N-(5-methylpyrazin-2-yl)-3-((R)-1-(triisopropylsilyloxy)propan-2-yloxy)propanamide(Intermediate AZ2) (0.595 g, 1.45 mmol) in anhydrous THF (20 mL) at 0°C. under nitrogen. The resulting solution was stirred at 0° C. for 10minutes and then3-chloro-2-(4-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)benzonitrile(Intermediate AH6) (0.438 g, 1.31 mmol) was added. The reaction mixturewas allowed to warm to room temperature and stirred for 1 hour. Thereaction mixture was adjusted to pH 7 by addition of 1M citric acid andthe majority of the THF removed in vacuo. The reaction mixture wasdiluted with water (20 mL) and EtOAc (50 mL). The organic layer wasseparated and the aqueous layer re-extracted with EtOAc (2×100 mL). Thecombined organics were washed with saturated brine (75 mL), dried(MgSO₄) and evaporated. The crude product was purified by flash silicachromatography, eluting with 50 to 100% EtOAc in isohexane to afford theproduct (0.590 g, 67.5%). ¹H NMR (400 MHz, CDCl₃) δ 0.98-1.08 (21H, m),1.12-1.17 (3H, m), 2.54 (3H, s), 3.59-3.78 (3H, m), 4.21-4.26 (1H, m),4.29-4.36 (1H, m), 6.02-6.06 (1H, m), 7.60-7.65 (1H, m), 7.78-7.81 (1H,m), 7.85-7.87 (1H, m), 8.11-8.13 (1H, m), 8.50 (1H, d), 8.61 (1H, d),8.71 (1H, d), 9.44 (1H, t); m/z (ES⁺) M⁺=665.26; HPLC t_(R)=4.02 min.

Intermediate AZ1:(2S)-2-[1-(2-chloro-6-cyanophenyl)pyrazolo[4,5-e]pyrimidin-4-yl]oxy-N-(5-methylpyrazin-2-yl)-3-[(2R)-1-tri(propan-2-yl)silyloxypropan-2-yl]oxypropanamide.Alternative preparation

Tris(dibenzylideneacetone)dipalladium(0) (0.261 g, 0.29 mmol) was addedin one portion to a de-gassed mixture of(2S)-2-(1-(2-bromo-6-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)-N-(5-methylpyrazin-2-yl)-3-((2R)-1-(triisopropylsilyloxy)propan-2-yloxy)propanamide(Intermediate BO1) (4.1 g, 5.70 mmol), zinc cyanide (0.335 g, 2.85 mmol)and 9,9-dimethyl-4,5-bis(diphenylphosphino)xanthene (0.330 g, 0.57 mmol)in N-methylpyrrolidone (41 mL) at 20° C. under nitrogen. The resultingsuspension was degassed under vacuum with inlet of nitrogen four timesand then heated to 110° C. (internal temperature) for 1 hour. Themixture was cooled, poured into water (100 mL) and extracted with ethylacetate (2×50 mL). The organic layers were combined and washed withwater (4×50 mL) and brine (50 mL), dried over MgSO₄, filtered andevaporated. The crude product was purified by flash silicachromatography, elution gradient 30 to 100% EtOAc in isohexane to affordthe product (3.0 g, 79%). ¹H NMR (400.13 MHz, DMSO-(d₆) δ 0.90-1.08(21H, m), 1.09-1.11 (3H, m), 2.45 (3H, s), 3.53-3.58 (1H, m), 3.60-3.66(1H, m), 3.75-3.78 (1H, m), 4.10-4.15 (2H, m), 5.94-5.99 (1H, m),7.87-7.91 (1H, m), 8.16-8.19 (2H, m), 8.31-8.33 (1H, m), 8.60-8.61 (1H,m), 8.73 (1H, s), 9.10-9.12 (1H, d), 11.20 (1H, s); m/z (ES⁻)(M−H)⁻=663; HPLC t_(R)=3.79 min.

Intermediate AZ1:(2S)-2-[1-(2-chloro-6-cyanophenyl)pyrazolo[4,5-e]pyrimidin-4-yl]oxy-N-(5-methylpyrazin-2-yl)-3-[(2R)-1-tri(propan-2-yl)silyloxypropan-2-yl]oxypropanamide.Alternative preparation

A solution of(2S)-2-(1-(2-bromo-6-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yloxy)-N-(5-methylpyrazin-2-yl)-3-((R)-1-(triisopropylsilyloxy)propan-2-yloxy)propanamide(Intermediate BO1) (1 equiv) in inerted* DMF (5 rel vols) anddiisopropylethylamine (DIPEA) (1.25 equivs) was kept in an inertatmosphere and was added with stirring to an inerted* mixture oftris(dibenzylideneacetone)dipalladium(0) (0.025 equivs), sodium acetate(0.1 equiv) and 1,1′-bis(diphenylphosphino)ferrocene (0.075 equivs) inDMF (5 rel vols) at 90° C. over 5 minutes. The mixture was stirred for10 minutes and then an inerted* solution of acetone cyanohydrin (1.4equivs) in DMF (2 rel vols) was added over 20 hours with stirring. Whenthe reaction was completed the mixture was cooled to 5° C. and dilutedwith methyl tert-butyl ether (7 rel vols) and then sodium bicarbonate(0.3 equivs) in water (2.5 rel vols). The organic phase was separatedand the aqueous phase washed with methyl tert-butyl ether. The combinedorganic extract and organic washings were evaporated under reducedpressure at 35° C. and the residue was purified by chromatography. Yield74%. *Nitrogen bubbled through before use.

Intermediate AZ2:(S)-2-hydroxy-N-(5-methylpyrazin-2-yl)-3-((R)-1-(triisopropylsilyloxy)propan-2-yloxy)propanamide

Trimethylaluminium (2M solution in toluene) (5.98 mL, 11.96 mmol) wasadded to 5-methylpyrazin-2-amine (1.305 g, 11.96 mmol) in toluene (20mL) cooled to 0° C. under nitrogen. The resulting solution was stirredat 0° C. for 20 minutes. (R)-methyl2-hydroxy-3-((S)-1-(triisopropylsilyloxy)propan-2-yloxy)propanoate(Intermediate AU5) (2 g, 5.98 mmol) in toluene (6 mL) was added and thereaction was allowed to warm to room temperature and then refluxed for 6hours. The reaction mixture was allowed to cool and concentrated invacuo. The residue was neutralised with citric acid (1M, aq.) and thendiluted with water (25 mL) and extracted with EtOAc (2×50 mL). Thecombined organics were washed with brine (20 mL), then dried (MgSO₄) andevaporated. The crude product was purified by flash silicachromatography, eluting with 30 to 50% EtOAc in isohexane to afford theproduct (1.390 g, 56.5%). ¹H NMR (400 MHz, CDCl₃) δ 1.04-1.17 (24H, m),2.54 (3H, s), 3.62-3.67 (3H, m), 3.81-3.85 (1H, m), 4.05-4.09 (1H, m),4.22 (1H, d), 4.34 (1H, q), 8.13 (1H, d), 9.12 (1H, s), 9.44 (1H, d);m/z (ES⁺) (M+H)⁺=412.34; HPLC t_(R)=3.82 min.

Intermediate AZ2:(S)-2-hydroxy-N-(5-methylpyrazin-2-yl)-3-((R)-1-(triisopropylsilyloxy)propan-2-yloxy)propanamide.Alternative preparation

Trimethylaluminium (2M solution in toluene) (72.5 mL, 145 mmol) wasadded to 5-methylpyrazin-2-amine (13.64 g, 125.0 mmol) in toluene (520mL) cooled to 0° C. under nitrogen. The resulting solution was stirredat 0° C. for 20 minutes. (R)-methyl2-hydroxy-3-((S)-1-(triisopropylsilyloxy)propan-2-yloxy)propanoate(Intermediate AU5) (33.5 g, 100.0 mmol) in toluene (130 mL) was addedand the reaction was allowed to warm to room temperature and thenstirred at 72° C. for 2.5 hours. The reaction mixture was allowed tocool then poured into a stirred mixture of ethyl acetate (500 mL) andaqueous solution of Rochelle salt (20% w/v, 500 mL). The mixture wasstirred for 15 minutes then the organics separated and washed with water(500 mL) followed by brine (500 mL), then dried (MgSO₄), filtered andconcentrated. The residues were dissolved in iso-hexane (300 mL) andstood for 15 minutes at 20° C. before cooling to 5° C. over 30 minutes.The solid formed was isolated by filtration, washed with iso-hexanecooled to 5° C. (200 mL) and dried at 40° C. under high vacuum to affordthe product (26.7 g, 64%). The filtrates were combined and concentratedto 100 mL total volume and the mixture stood for 16 hours. The solidformed was isolated by filtration, washed with iso-hexane cooled to 5°C. (100 mL), dried at 40° C. under high vacuum and combined with theinitial batch to afford the product (30.1 g, 73%). ¹H NMR (400 MHz,CDCl₃) δ 1.04-1.17 (24H, m), 2.54 (3H, s), 3.62-3.67 (3H, m), 3.81-3.85(1H, m), 4.05-4.09 (1H, m), 4.22 (1H, d), 4.34 (1H, q), 8.13 (1H, d),9.12 (1H, s), 9.44 (1H, d); m/z (ES⁺) (M+H)⁺=412.34; HPLC t_(R)=3.82min.

Intermediate BO1:(2S)-2-[1-(2-bromo-6-chlorophenyl)pyrazolo[4,5-e]pyrimidin-4-yl]oxy-N-(5-methylpyrazin-2-yl)-3-[(2R)-1-tri(propan-2-yl)silyloxypropan-2-yl]oxypropanamide

Sodium hydride (0.875 g, 21.87 mmol) was added to(S)-2-hydroxy-N-(5-methylpyrazin-2-yl)-3-((R)-1-(triisopropylsilyloxy)propan-2-yloxy)propanamide(Intermediate AZ2) (3.0 g, 7.29 mmol) in anhydrous THF (35 mL) at 0° C.under nitrogen. The resulting solution was stirred at 0° C. for 10minutes and then a solution of1-(2-bromo-6-chlorophenyl)-4-chloro-1H-pyrazolo[3,4-d]pyrimidine(Intermediate BO2) (2.507 g, 7.29 mmol) in anhydrous THF (20 mL) wasadded dropwise over 1 minute. The mixture was stirred at 0° C. for 10minutes and then allowed to warm to ambient temperature for 6 hours. Itwas then cooled to ˜0° C., the mixture poured into ice cold 1M citricacid and diluted with EtOAc (50 mL). It was stirred vigorously for 10minutes. The organic phase was separated, washed with water and brine,dried over MgSO₄, filtered and evaporated. The crude product waspurified by flash silica chromatography, elution gradient 20 to 50%EtOAc in isohexane to afford the product (4.90 g, 93%).

¹H NMR (400.13 MHz, DMSO-d₆) δ 0.88-1.03 (21H, m), 1.09-1.11 (3H, m),2.44 (3H, s), 3.52-3.57 (1H, m), 3.63-3.67 (1H, m), 3.73-3.78 (1H, m),4.09-4.18 (2H, m), 5.92-5.98 (1H, m), 7.60-7.64 (1H, m), 7.80-7.83 (1H,m), 7.91-7.93 (1H, m), 8.30-8.31 (1H, m), 8.53-8.54 (1H, m), 8.63 (1H,s), 9.11 (1H, s), 11.19 (1H, s); m/z (ES⁻) (M−H)⁻=718; HPLC t_(R)=3.95min.

Intermediate BO1:(2S)-2-[1-(2-bromo-6-chlorophenyl)pyrazolo[4,5-e]pyrimidin-4-yl]oxy-N-(5-methylpyrazin-2-yl)-3-[(2R)-1-tri(propan-2-yl)silyloxypropan-2-yl]oxypropanamide.Alternative method

A solution of1-(2-bromo-6-chlorophenyl)-4-chloro-1H-pyrazolo[3,4-d]pyrimidine(Intermediate BO2) (1.03 equivs) in toluene (8 rel vols) and THF (2 relvols) was added to a stiffed mixture containing an aqueous sodiumhydroxide solution (4 equivs) and(S)-2-hydroxy-N-(5-methylpyrazin-2-yl)-3-(R)-1-(triisopropylsilyloxy)propan-2-yloxy)propanamide(Intermediate AZ2) (1.00 equiv) in toluene (2 rel vols) and THF (5 relvols) at 10° C. The mixture was allowed to warm to ambient temperatureand stirred at ambient temperature until full conversion (1 hour). Thereaction mixture was added to a cold mixture of citric acid (3 equivs),water (4.5 rel vols) and toluene (8 rel vols) with stiffing. The mixturewas allowed to warm up to ambient temperature with stirring. The mixturewas separated and the organic phase was washed with brine andconcentrated. The product is obtained by evaporation of the solvent.Yield 94%.

Intermediate BO2:1-(2-bromo-6-chlorophenyl)-4-chloropyrazolo[4,5-e]pyrimidine

Phosphoryl trichloride (30.4 ml, 326.37 mmol) was added in one portionto 1-(2-bromo-6-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-ol(Intermediate AH4) (4.25 g, 13.05 mmol) and the resulting suspension wasstirred at 100° C. for 2 hours.

It was then cooled and evaporated. The residue was dissolved in toluene(60 mL), poured onto ice, diluted with ethyl acetate (40 mL) and stirredfor 1 hour. The organic phase was separated, washed with water (4×25 mL)and brine (25 mL), dried over MgSO₄, filtered and evaporated to a paleyellow oil that crystallised on standing to afford the product (4.3 g,95%) which was used without further purification.

¹H NMR (400.13 MHz, DMSO-d₆) δ 7.66 (1H, dd), 7.84 (1H, dd), 7.94 (1H,dd), 8.89 (1H, s), 8.93 (1H, s); m/z (ES⁺) (M+H)⁺=345; HPLC t_(R)=2.90min.

Intermediate BO2:1-(2-bromo-6-chlorophenyl)-4-chloropyrazolo[4,5-e]pyrimidine.Alternative procedure

Phosphoryl trichloride (4 equivs) was added to a slurry of1-(2-bromo-6-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-ol(Intermediate AH4) (1 equiv) in acetonitrile (3 rel vols) with stirringunder nitrogen followed by diisopropylethylamine (4 equivs). Thereaction mixture was boiled under reflux with stirring for 15 hours. Themixture was cooled to ambient temperature and toluene (12 rel vols) wasadded. The mixture was cooled at 1° C. and quenched with water (3 relvols) keeping the temperature below 20° C. The organic layer wasseparated off and washed with water, then with aqueous sodiumbicarbonate solution then dried to give a solution of the product intoluene. Yield 94%.

BIOLOGICAL Tests:

The biological effects of the compound of the invention may be tested inthe following way:

(1) Enzymatic Activity

Enzymatic activity of recombinant human pancreatic GLK may be measuredby incubating GLK, ATP and glucose. The rate of product formation may bedetermined by coupling the assay to a G-6-P dehydrogenase, NADP/NADPHsystem and measuring the linear increase with time of optical density at340 nm as described in Brocklehurst et al (Diabetes 2004, 53, 535-541).

The compound of the invention has been found to have the following meanEC₅₀ (μM):

Ex. No. EC50/uM 1 0.4415

Production of Recombinant GLK:

Human GLK was obtained by PCR from human pancreatic mRNA respectively,using established techniques described in Sambrook J, Fritsch E F &Maniatis T, 1989. PCR primers were designed according to the GLK cDNAsequences shown in Tanizawa et al 1991 and Bonthron, D. T. et al 1994(later corrected in Warner, J. P. 1995).

Cloning in Bluescript II vectors

GLK was cloned in E. coli using pBluescript II, (Short et al 1998) arecombinant cloning vector system similar to that employed byYanisch-Perron C et al (1985), comprising a colEI-based replicon bearinga polylinker DNA fragment containing multiple unique restriction sites,flanked by bacteriophage T3 and T7 promoter sequences; a filamentousphage origin of replication and an ampicillin drug resistance markergene.

Transformations

E. Coli transformations were generally carried out by electroporation.400 mL cultures of strains DH5a or BL21(DE3) were grown in L-broth to anOD 600 of 0.5 and harvested by centrifugation at 2,000 g. The cells werewashed twice in ice-cold deionised water, resuspended in 1 mL 10%glycerol and stored in aliquots at −70° C. Ligation mixes were desaltedusing Millipore V series™ membranes (0.0025 mm) pore size). 40 mL ofcells were incubated with 1 mL of ligation mix or plasmid DNA on ice for10 minutes in 0.2 cm electroporation cuvettes, and then pulsed using aGene Pulser™ apparatus (BioRad) at 0.5 kVcm⁻¹, 250 mF. Transformantswere selected on L-agar supplemented with tetracyline at 10 mg/mL orampicillin at 100 mg/mL.

Expression

GLK was expressed from the vector pTB375NBSE in E. coli BL21 cells,producing a recombinant protein containing a 6-His tag immediatelyadjacent to the N-terminal methionine. Alternatively, another suitablevector is pET21(+)DNA, Novagen, Cat number 697703. The 6-His tag wasused to allow purification of the recombinant protein on a column packedwith nickel-nitrilotriacetic acid agarose purchased from Qiagen (cat no30250).

(2) Oral Glucose Tolerance Test (OGTT) or Glucose Profile

Oral glucose tolerance tests were done on conscious Zucker obese fa/farats (age 12-13 weeks or older). The animals were fasted for 2 hoursbefore use for experiments. A test compound or a vehicle was givenorally 120 minutes before oral administration of a glucose solution at adose of 2 g/kg body weight. Blood glucose levels were measured using aAccucheck glucometer from tail bled samples taken at different timepoints before and after administration of glucose (time course of 60minutes). A time curve of the blood glucose levels was generated and thearea-under-the-curve (AUC) for 120 minutes was calculated (the time ofglucose administration being time zero). Percent reduction in glucoseexcursion was determined using the AUC in the vehicle-control group aszero percent reduction.

For Glucose profile a test compound or vehicle was given 60 minutesbefore conscious Zucker obese fa/fa rats (age 12-13 weeks or older)entered a dark cycle (12-hours). Blood glucose mevels were measuredusing a Accucheck glucometer from tail bled samples taken at differenttime points during the 12-hour dark cycle. A time curve of the bloodglucose levels was generated and the area-under-the-curve (AUC) for12-hours was calculated (the beginning of the dark cycle being timezero). Percent reduction in glucose excursion was determined using theAUC in the vehicle-control group as zero percent reduction.

1:(2S)-2-[1-(2-Chloro-6-cyano-phenyl)pyrazolo[4,5-e]pyrimidin-4-yl]oxy-3-[(1R)-2-hydroxy-1-methyl-ethoxy]-N-(5-methylpyrazin-2-yl)propanamideor a pharmaceutically acceptable salt thereof. 2: The compound asclaimed in claim 1 which is(2S)-2-[1-(2-chloro-6-cyano-phenyl)pyrazolo[4,5-e]pyrimidin-4-yl]oxy-3-[(1R)-2-hydroxy-1-methyl-ethoxy]-N-(5-methylpyrazin-2-yl)propanamide.3: The compound as claimed in either claim 1 or claim 2 in crystallineform. 4: A pharmaceutical composition comprising a compound as claimedin either claim 1 or claim 2 together with a pharmaceutically acceptablediluent or carrier. 5-6. (canceled) 7: A method of treating a GLKmediated disease by administering an effective amount of a compound asclaimed in either claim 1 or claim 2 to a mammal in need of suchtreatment. 8: The method of claim 7 wherein the GLK mediated disease istype 2 diabetes. 9-10. (canceled) 11: A pharmaceutical combinationcomprising a compound as claimed in either claim 1 or claim 2 andanother pharmacologically active substance. 12: The pharmaceuticalcombination as claimed in claim 11 wherein the other pharmacologicallyactive substance is a medicament for the treatment of type 2 diabetes.13: The method of claim 7 wherein the disease mediated through GLK isobesity. 14: A process for the preparation of(2S)-2-[1-(2-chloro-6-cyano-phenyl)pyrazolo[4,5-e]pyrimidin-4-yl]oxy-3-[(1R)-2-hydroxy-1-methyl-ethoxy]-N-(5-methylpyrazin-2-yl)propanamidecomprising de-protecting a compound of formula A

in which Prot represents a hydroxy protecting group. 15: A compound offormula A

in which Prot represents a hydroxy protecting group.